- Volume 140, Issue 4, 1994

One problem of peptide vaccines is that antibodies generated against them react poorly with the target sequence on the native protein. Using monoclonal antibodies (mAbs) to the serovar L1 type-specific epitope on the major outer-membrane protein of Chlamydia trachomatis as our model in conjunction with the Pin Technology Epitope Scanning technique, we had previously identified the critical binding site at this epitope as DAVP. Amino acid substitution showed that AV were essential residues for binding. A series of structurally related (heteroclitic) peptides retaining AV were synthesized. Some of these were found to be much more reactive with the model mAb than peptides of cognate sequence. It was hypothesized that the DAVP peptide only approximated to the conformation of the homologous sequence in the native protein, whereas some of the flexible heteroclitic peptides produced conformations which more closely resembled the native constrained sequence. The key question was whether the most reactive heteroclitic peptide would also generate antibody capable of more efficient binding to the native protein. We therefore immunized mice with one of six heteroclitic peptides or one of two native sequence control peptides. The reactivity of these antisera with the peptide immunogens and with native chlamydial elementary bodies was then evaluated by enzyme immunoassay. Pooled antisera to two of the heteroclitic peptides reacted with significantly greater absorbance (P < 0·05) and at higher dilution with whole chlamydiae than did pooled antisera to the control peptides. This suggests that heteroclitic peptides may in some circumstances be useful to increase the reactivity of site-specific antibodies with epitopes on the native protein important for vaccine development or for serodiagnosis.

Pediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61, was purified and partially characterized. The purification was achieved by dialysis against PEG 20000, butanol extraction and electroendosmotic preparative electrophoresis. The protocol led to a 7843-fold increase in the specific activity, with 3·9% activity recovered. SDS-PAGE of pediocin A resulted in a single 80 kDa protein band. The antimicrobial compound was sensitive to proteolytic enzymes and heat (10 min at 100 °C). It exhibited inhibition against species of Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Staphylococcus, Enterococcus, Listeria and Clostridium.

An inducible ferulic acid esterase (FAE-III) has been isolated, purified and partially characterized from Aspergillus niger after growth on oat spelt xylan. The purification procedure utilized ammonium sulphate precipitation, hydrophobic interaction and anion-exchange chromatography. The purified enzyme appeared almost pure by SDS-PAGE, with an apparent M r of 36000. A single band, corresponding to a pl of 3·3 was observed on isoelectric focusing. With methyl ferulate as substrate, the enzyme had a specific activity of 67 IU (mg protein)−1, pH and temperature optima of 5 and 55–60 °C, respectively, and a Km of 2·08 mM and a V max of 175 μmol min−1 (mg protein)−1. The enzyme was also active upon methyl sinapinate, methyl-3,4-dimethoxy cinnamate and methyl p-coumarate, but not benzoic acid methyl esters or methyl caffeate. Similarly, Streptomyces olivochromogenes FAE showed activity against methyl ferulate, methyl sinapinate and methyl p-coumarate, but at a level 420-fold less (on methyl ferulate) than the A. niger esterase. No activity was detected against the benzoate methyl esters. For both enzymes, this shows the necessity for C-3 on the phenol ring to be methoxylated and the aliphatic region of the substrate to be unsaturated. The specific activity of FAE-III on destarched wheat bran was 31 U (mg protein)−1 in the presence of Trichoderma viride xylanase and 3 U (mg protein)−1 in the absence. Apparent pH dependent binding of A. niger FAE-III to microcrystalline cellulose was also demonstrated.

Chorismate pyruvate-lyase from Escherichia coli converts chorismate to 4-hydroxybenzoate. The enzyme was enriched 3000-fold by overexpression and chromatographic purification. It has an apparent K m value for chorismate of 6.1 μM and an isoelectric point of pH 6.45. The enzyme activity did not require metal cofactors. Promoter sequences in the 5′ flanking sequences of the ubiCA operon were localized by transcription and translation of active chorismate pyruvate-lyase in vitro from different PCR fragments. Sequencing of the ubiC gene of the mutant strain AN244 revealed a G→A transition resulting in a change from glutamic acid to lysine. A feeding experiment with [1,7-13C2]shikimate confirmed the chorismate pyruvate-lyase as the sole enzymic source of 4-hydroxybenzoate in vivo.

Bacteriophage A7 has been employed as an indicator for strains of Pseudomonas syringae pv. morsprunorum race 1. Electron microscopy showed that this phage had a hexagonal head, 59 nm in diameter, and a long, flexible, non-contractile tail, 164 nm long and 8 nm wide, containing approximately 15 evenly spaced transverse striations and terminating in a base-plate equipped with six spikes arranged in a radially symmetrical pattern around a central core. Two short fibres projecting at an angle from the base-plate were also visible on some phage particles. Phage A7 can therefore be placed in group B of the classification system of Bradley. Phage particles bound at apparently random sites over the surface of host cells by their base-plates, and after a short time released DNA from their heads. Phage A7 uses lipopolysaccharide (LPS) as its binding site on the bacterial cell surface, removing the d-rhamnan side-chains by the action of a rhamnan hydrolase. The appearance of purified LPS by electron microscopy was either strand-like or vesicular, according to whether it had been stained with phosphotungstate or uranyl acetate respectively. Strands were of approximately uniform width (approx. 9 nm). Vesicular forms included both circles, 25–45 nm in diameter, and larger oval structures of greater electron transparency. The appearance of the LPS did not alter on addition of the phage. Phage particles were observed attached via their base-plates to LPS vesicles, in particular the larger oval vesicles, but were never seen attached to strands. The heads of phage particles attached to LPS were frequently empty. The rhamnanase of phage A7 released the side-chain residues from the LPS as an equimolar mixture of tri- and hexasaccharide. By using 1 h-NMR spectroscopy and methylation analysis, the site of cleavage has been identified as the 2)-α-d-Rhap(1 → 3) residue.

An endopeptidase has been purified from Lactococcus lactis subsp. cremoris SK11. The enzyme is a 70 kDa monomer, strongly inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and phosphoramidon but relatively insensitive to EDTA. It is not significantly inhibited by the thiol enzyme inhibitor p-chloromercuribenzoate nor by the serine protease inhibitor phenylmethylsulphonyl fluoride. The action of the endopeptidase in catalysing the hydrolysis of several peptide hormones has been studied and the hydrolysis products identified by sequence analysis. The enzyme catalyses hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly a Phe or Leu) residue occupies the position immediately C-terminal to the hydrolysed bond. It thus has a specificity very similar to that of thermolysin. Two of the oligopeptides produced during the early stages of β-casein digestion by the lactococcal cell-wall proteinases were hydrolysed by the endopeptidase, the others were resistant to hydrolysis. Cell fractionation studies have shown that the distribution of endopeptidase activity between the different cell fractions is the same as that of the intracellular marker enzyme fructose bisphosphate aldolase, and thus indicate a cytoplasmic location for the enzyme. These observations argue against a role for this enzyme in the early stages of casein breakdown by the lactococcal proteolytic system.

A clinical isolate of Aeromonas hydrophila was demonstrated to transport [14C]maltose with similar kinetics to enteric bacteria (K m: 0.3μM; V max: 22 nmol min−1 per 109 cells). The uptake of [14C]maltose was completely inhibited in the presence of unlabelled maltose or maltodextrins, whereas other mono- and disaccharides, such as glucose, galactose, sucrose, lactose or melibiose, had no effect. A protein with an apparent molecular mass of 39 kDa (maltose-binding protein; MBP) was identified in osmotic-shock fluid of maltose-grown cells by SDS-gel electrophoresis, and was purified to homogeneity by either amylose affinity chromatography or ion-exchange chromatography. Equilibrium dialysis experiments revealed the ability of the purified protein to bind [14C]maltose with high affinity (KD = 1.6μM). Unlabelled maltose and maltodextrins competed for the binding site. In a reconstitution experiment, A. hydrophila MBP poorly restored the transport activity of a binding-protein-deficient Escherichia coli (ΔmalE) mutant. N-terminal sequence analyses of the purified native protein and of peptides generated by cleavage with CNBr and subsequently separated by HPLC revealed about 56% identical amino acid residues, as compared to enterobacterial MBPs. We conclude that maltose is transported into A. hydrophila via a binding-protein-dependent transport system.

To better understand the reasons for the hyper-accumulation of polyhydroxyalkanoate (PHA) in mutant Azotobacter vinelandii UWD, the kinetic properties of 3-ketothiolase, acetoacetyl-CoA reductase, and β-hydroxybutyrate dehydrogenase were examined. The regulation of the condensation of acetyl-CoA mediated by 3-ketothiolase was normal, in that it was negatively regulated by free CoA, but inhibition was overcome by higher concentrations of acetyl-CoA. Acetoacetyl-CoA from this reaction was reduced to 3-hydroxybutyryl-CoA by an NADPH-specific acetoacetyl-CoA reductase. This enzyme also reduced 3-ketovaleryl-CoA derived from the β-oxidation of C5, C7 or C9 n-alkanoates, but at only 16% of the rate found with the C4-substrate. The acetoacetyl-CoA reductase was determined to be an allosteric enzyme that bound NADPH and acetoacetyl-CoA at multiple binding sites in a general hybrid Ping-Pong random mechanism. The enzyme was negatively regulated by acetoacetyl-CoA, but this was overcome at high concentrations of NADPH. The activity of pyridine nucleotide transhydrogenase was determined to be important for the conversion of NADH in these mutant cells to NADPH and for decreasing the availability of NADP+, which was a negative regulator of the acetoacetyl-CoA reductase. The combination of high acetoacetyl-CoA, the UWD mutation, transhydrogenase activity, and high NADPH appeared to be the conditions promoting PHA formation by strain UWD during active growth on glucose. Degradation of PHA in strain UWD did not appear to be regulated at the level of β-hydroxybutyrate dehydrogenase. This enzyme was unaffected by NADH, was inhibited only 13% by pyruvate and its activity was enhanced by NADPH. The thiolysis of acetoacetyl-CoA also was unusual, in that 3-ketothiolase was not inhibited by acetoacetyl-CoA, but free CoA was a competitive inhibitor in a bireactant Ping-Pong mechanism. This inhibition was overcome by higher concentrations of the normal first substrate, acetoacetyl-CoA. Thus a single thiolase was used for the condensation of acetyl-CoA and the thiolysis of acetoacetyl-CoA, derived from PHA depolymerization or from the β-oxidation of n-alkanoates.

l-Glutamate and l-aspartate are the natural nitrogen sources preferred by the fungus Pleurotus ostreatus. In the vegetative mycelium NAD-dependent glutamate dehydrogenase (NAD-GDH) and glutamine synthetase (GS) were both present, but the highest level of these enzymes was found in the fruiting body. NAD-GDH was derepressed by ammonia and repressed by higher concentrations of l-glutamate. Otherwise, the level of the enzyme in mycelium was independent of the nitrogen source. GS was derepressed by ammonia, l-glutamate, l-aspartate and l-alanine. Repression of GS was observed in the presence of l-glutamine, l-arginine, nitrate, urea and low concentrations of ammonia. An increase in the ratio of glucose to nitrogen resulted in a decrease in the level of GS in the mycelium. The K m values of NAD-GDH for 2-oxoglutarate, ammonia, NADH, l-glutamate and NAD+ were 2·9 mM, 3·3 mM, 11 μM, 0·18 mM and 62 μM, respectively. The K m values of GS for l-glutamate and ammonia were 2·5 and 0·27 mM, respectively. The repression of GS and derepression of NAD-GDH in the presence of low concentrations of ammonia together with glutamate or aspartate could be explained by an inhibition or repression of some amino acid permeases by ammonia. The absence of NADP-dependent glutamate dehydrogenase and glutamate synthase together with the apparent mode of regulation of NAD-GDH and GS synthesis suggest that P. ostreatus assimilates ammonia via GS and NAD-GDH. However, a dissimilatory role of NAD-GDH in the deamination of l-glutamate, due to its very low K m for l-glutamate, is not excluded.

Membranes obtained from whole-cell lysates of Burkholderia (Pseudomonas) pseudomallei (strain 319a) were separated into four fractions by sucrose density gradient centrifugation. Membranes were characterized by enzymic and chemical analyses, and by SDS-PAGE. Cytoplasmic membranes and two forms of outer membranes (OM-1, OM-2) were detected. The major outer-membrane proteins had M r values of 70000, 38000, 31000, 24000 and 17000. To determine which outer-membrane proteins were common to B. pseudomallei strains, OM-1 fractions from 12 different strains were prepared. SDS-PAGE analysis of these fractions demonstrated that the five major outer-membrane proteins were common to the strains tested. Further studies have shown that an M r 110000 protein, which is oligomeric in that it migrates as an M r 38000 protein upon heating at 95 °C and which is peptidoglycan-associated, serves as a porin in B. pseudomallei. Using proteoliposomes reconstituted from this protein and phospholipid, it was demonstrated by the liposome-swelling assay that this protein acts as a porin through which small saccharides may diffuse. Further characterization of this M r 38000 protein will be important in delineating the role of this molecule in the permeability of the B. pseudomallei outer membrane.

The cloning of the Bacillus subtilis glpT and glpQ genes and their nucleotide sequences are reported. Analysis of mRNA indicates that glpT and glpQ constitute one operon which is transcribed from a sigma A type promoter. The steady state amount of glpTQ mRNA is increased in cells grown in the presence of glycerol 3-phosphate. The 5′ untranslated leader sequence of glpTQ mRNA contains an inverted repeat which shows sequence similarity to repeats present in the leader sequences of glpFK and glpD transcripts. These repeats seem therefore to be essential control elements for all B. subtilis glp genes.

The Bacillus subtilis hemAXCDBL operon encodes enzymes for the biosynthesis of uroporphyrinogen III from glutamyl-tRNA. The function of the hemX gene product was studied in this work. The deduced amino acid sequence suggests HemX to be an integral 32 kDa membrane protein. This was confirmed by experiments using Escherichia coli minicells and hemX-phoA gene fusions. Deletion of the hemX gene from the Bacillus subtilis chromosome demonstrated that this gene is not required for haem synthesis. However, the deletion strain was found to overexpress the hemA gene product, glutamyl-tRNA reductase. A combination of results obtained with B. subtilis hemA and hemX in Escherichia coli and Bacillus subtilis shows that HemX negatively affects the steady-state cellular concentration of HemA protein. The mechanism by which HemX affects the HemA concentration is unclear.

Genes specifically expressed in the parasitic yeast phase of Histoplasma capsulatum have been cloned to clarify the mechanisms underlying both pathogenesis and morphogenesis in this human dimorphic fungal pathogen. Previous studies have determined that the yeast-phase-specific gene, yps-3, is expressed differentially in two non-isogenic strains which differ in their thermotolerance and virulence. We have cloned the yps-3 homologues from the high virulence (G217B) and low virulence (Downs) strains, and obtained a partial cDNA clone representing the expressed gene from H. capsulatum G217B. The Downs clone harbours a 287 bp insertion sequence that disrupts a long ORF defined by the yps-3 G217B cDNA. Although the insertion sequence contains features reminiscent of mobile genetic elements, including 15 bp direct repeats of flanking sequence, it is not randomly distributed in the H. capsulatum genome. S1 nuclease analysis was utilized to map the 5′ end of the expressed yps-3 gene in G217B to potential regulatory regions which are largely homologous in both strains. This finding may point to a deficiency in a temperature inducible regulatory protein in the low virulence, temperature-sensitive Downs strain. The nucleotide sequence of the yps-3 gene and the predicted amino acid sequence of its product represents the first report of phase-specific gene and protein sequences in this widely distributed fungal pathogen. Further analysis of the product encoded by the yps-3 gene may provide significant insight into the pathogenic repertoire of H. capsulatum.

The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae and the fnr gene of Escherichia coli encode very similar proteins. The hlyX gene is able to complement Δfnr mutations and will permit the growth of E. coli fnr strains in nitrate minimal salts medium under anoxic conditions. In addition, the hlyX gene product appears to induce the expression of a latent haemolytic activity as evidenced by the presence of a strong zone of haemolysis around E. coli (hlyX +) colonies grown on bovine or ovine blood. In this study, the ability of the hlyX gene product to induce haemolytic activity and regulate expression of frdA and its own gene was examined in the presence of various carbon sources and in the presence and absence of iron; fnr was included for comparison. The HlyX protein was able to induce the synthesis of the latent E. coli haemolytic activity only under anoxic conditions. Haemolytic activity was highest during the late exponential phase and then levelled off in the stationary phase. The hlyX gene product was able to activate the expression of a ø (frdA’-lacZ) in E. coli JRG1787 (Δfnr); however, the level of expression depended on carbon source, growth phase and copy number. Like fnr, the hlyX gene product appeared to affect its own synthesis but the nature and extent of regulation depended not only on the presence of oxygen but also on growth conditions.

Monohaem cytochrome c-553 from Desulfovibrio vulgaris Hildenborough is encoded by the cyf gene which could be expressed in Escherichia coli to yield the periplasmic holoform of cytochrome c-553. Covalent haem attachment was shown by labelling with 5-amino[4-14C]laevulinic acid, the immediate precursor for haem biosynthesis. Visible-absorption spectroscopy demonstrated that the haem environment in the recombinant protein did not differ from the native protein. Optimal expression was obtained under aerobic conditions. Furthermore, efficient insertion of haem into a cytochrome c-553-β-lactamase fusion protein could be demonstrated. We suggest that the varying success in heterologous expression of c-type cytochromes in E. coli may arise as a result of differences in the physical properties of the apoproteins.

The gene of Proteus mirabilis coding for a single-stranded DNA-binding protein (SSB) was cloned in Escherichia coli from a genomic library. It restored the UV resistance and the rate of cell division of an E. coli ssb-113 mutant to the same extent as the cloned E. coli ssb gene did. An E. coli mutant with deleted ssb was viable with the P. mirabilis ssb + gene provided on a single-copy-number plasmid and had the same cell division rate as with the E. coli ssb + gene on the same vector plasmid. The recovery from UV damage of an excision repair deficient (uvrA) mutant deleted for the ssb gene was identical with the ssb + gene from P. mirabilis or E. coli, suggesting full substitution in recombinational DNA repair of the homologous by the heterologous SSB protein. The nucleotide sequence of the gene revealed that the SSB has 81% amino acid sequence homology with the E. coli SSB and only 58–63% with various plasmid SSBs. The data provide evidence that the bacterial chromosomally coded SSBs and the plasmid encoded SSBs constitute separate groups.

The CAM (camphor degradation) plasmid is integrated into the chromosome of Pseudomonas putida PaW-line strains and is not selftransferable as a plasmid via conjugation. Our results show that the mobilization of chromosomally located CAM and the integration of cam-operons into the chromosome of the new Cam+ transconjugants is a recA-independent process mediated by transposons Tn4652 (17 kbp) and Tn3614 (7.2 kbp). Transposon Tn3614 is apparently identical to the left-hand and the right-hand sequences of the TOL plasmid pWWO transposon Tn4654. The insertion of Tn401 inside the left-hand terminal IR of Tn4652 completely inhibited the mobilization of CAM. According to our data transposons Tn4652 and Tn3614 together with CAM plasmid catabolic operons are integrated into the chromosome. We propose that in eseudomonads the transposons Tn4652 and Tn3614 play a key role in the evolution and spread of new catabolic plasmids in nature.

We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-1 gene encodes a protein with 71 % amino acid residues identical to the well characterized elongation factor Tu of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-1 downstream of a fus homologue and the in vitro activity of Sr. EF-Tu1 show that tuf-1 encodes a genuine EF-Tu. The putative Sr. EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EF-Tu. Homologues of tuf-1 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tu1 was present at all times under kirromycin production conditions in submerged and surface-grown cultures of S. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tu1 was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of S. ramocissimus.